The present invention is directed to a self-quenching primer and its use in amplification reactions, particularly in polymerase chain reactions, during which the fluorophore is released thereby emitting fluorescence.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
PCR products can be quantitated during the linear portion of amplification allowing an accurate quantitation of templates. There have been many quantitation methods developed based on PCR amplification. Several of these are summarized below.
Real-time PCR has been the most widely used in which PCR products are monitored in real time mainly through fluorescence emitted in association with PCR products. The several approaches to generate fluorescence in association with PCR products include the use of nucleic acid dyes (e.g. SYBR Green I) and Fluorescence Resonance Energy Transfer (FRET).
Dye-based methods are comparatively simple because specific binding of certain dyes, such as SYBR green I, to double strand DNA will emit fluorescence (FIG. 1). Such methods, however, are not as specific. Since the fluorescence depends solely on the amount of ds DNA, which includes specific products, non-specific products and primer dimers, it is not specific to a particular PCR product.
Fluorescence Resonance Energy Transfer (FRET) (Clegg, 1992) refers to a process by which energy is transferred from one dye molecule (the donor) to another (the acceptor) without the emission of a photon. FRET technology has been used in several ways to develop real time hybridization assays including the Roche FRET assay, TaqMan® assay, molecular beacon and their derivatives. In FRET, if the acceptor dye is a fluorophore, the energy may be emitted as fluorescence that is characteristic of the acceptor dye, otherwise the energy is dissipated and the fluorescence quenched. The Roche FRET assay (FIG. 2) uses two oligonucleotides probes with one carrying the donor and the other one carrying the receptor molecule at their adjacent ends. The binding to the PCR products by the two oligonucleotides puts the two fluorescent dyes close to each other; thereby the acceptor dye will emit fluorescence upon accepting energy from the donor. Fluorescent detection is conducted during the annealing step. This technique is very specific since emission of fluorescence from receptor depends not only on the PCR products but also on specific binding to the PCR products by the two primers. However, it is sometimes difficult to design four primers for one target sequence, especially with two primers adjacent to each other. It is not suitable for short target sequences such as transgenic elements in highly degraded DNA samples. The cost is also a concern since two primers each labeled with one fluorescent dye are required for each target sequence.
Another technique, the TaqMan® assay (Livak et al., 1995), also uses FRET to monitor PCR reactions in real time. It needs two primers and one probe for a target sequence (FIG. 3). The probe is an oligonucleotide complementary to a region between the forward and reverse primer with the fluorescent donor and quenching receptor dye attached to its 5′ and 3′ ends. The energy transfer from the fluorescent donor to receptor dye will quench fluorescence. This probe will bind to PCR fragment but will later be deleted by DNA polymerase with 5′ exonuclease activity. Degradation of the probe will separate the donor from the receptor molecule and hence the donor molecule will emit fluorescence. The TaqMan® assay is as specific as the Roche FRET assay, but requires that the three oligonucleotides be close to each other, an optimal distance between the forward primer and the probe of less than 10 bp while the distance of the probe from the reverse primer as short as possible. Tm for the probe should also be higher than those of forward and reserve primers, preferably by 8° to 10° C. In this method, there is still the need to use two fluorescent dyes for each target sequence.
A derivative of the TaqMan® assay, UT-PCR (Zhang et al., 2003), uses a universal oligonucleotide with two dyes attached at two ends with fluorescent donor quenched by the non-fluorescent quencher. The forward primer has the complementary sequence (universal template) to this oligonucleotide attached to its 5′ end. The oligonucleotide will bind to 5′ universal sequence of forward primer during annealing. At the end of DNA polymerization in the reverse direction, the oligonucleotide with two dyes will be deleted, releasing the fluorescent donor. This design uses only two specific primers, compatible with general PCR. The main advantage is the potential cost saving by using dyes attached to a universal oligonucleotide. However, it may have the problem of non-linear increase of fluorescence due to competition between the universal oligonucleotide and PCR product for forward primer.
A molecular beacon (FIG. 4) is made up of an oligonucleotide with a fluorescent dye attached to one end and a quencher (non-fluorescent acceptor dye) attached to the other. The sequence is designed so that the oligonucleotide forms a hairpin loop, which brings the fluorescent dye and quencher together. In this configuration, the fluorescence is nearly completely quenched in solution. The loop portion of the hairpin is complementary to the sequence of interest and between the forward and reverse primer. Once hybridization to the sequence on template or PCR products, the hairpin unfolds, separating the fluorescent dye from the quencher. Thus, a fluorescent signal indicates hybridization of the molecular beacon to the sequence of interest and its intensity correlate to the quantity of PCR products. This technique has lower background than others but still requires a tailor made third primer with two dyes. It also has some requirement for the loop portion of the hairpin.
BODIPY® FL developed by Molecular Probes is 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid. Its structure is shown in FIG. 5. It has several characteristics that make it potentially possibility in many applications. These include: high extinction coefficient; high fluorescence quantum yield; spectra that are relatively insensitive to solvent polarity and pH; narrow emission bandwidth which resulting in a higher peak intensity than that of fluorescein; relatively long excited-state lifetimes, making the dyes useful for fluorescence polarization-based assays; little or no spectral overlap with longer-wavelength dyes which making it one of the preferred green-fluorescent dyes for multicolor application; and lack of ionic charge.
BODIPY® FL can be attached to an oligo at 5′ by a linker. Horn et al. (1997) reported that the fluorescent emission from a probe modified with fluorophore BODIPY® FL was diminished after hybridization. Kurata et al. (2001) found that the quenching was caused by the interaction between the fluorophore and a guanine base. Furthermore, it is reported that fluorescence intensity decreases much more when guanine is opposite to the fluorophore than at another position. Kurata et al. (2001) used this discovery to design an oligonucleotide probe for the quantitative detection of target DNA. Kurata et al. (2001) also modified a primer by adding a cytosine and BODIPY® FL and used this primer for real-time quantitative PCR. It was found that a guanine was added to the primer during PCR resulting in the quenching of fluorescence. The initial quantity of target present in the sample was determined by utilizing a fluorescence quench rate.
Also making use of this discovery, Tani et al. (2005) designed an oligonucleotide with BODIPY® FL attached to its 3′ end that is complementary to a sequence between the forward and the reverse primer. Binding to this oligonucleotide will put the BODIPY® FL just opposite guanines on template DNA and hence its fluorescence quenched. It was found that the decrease of fluorescence when detected after annealing was proportional to the PCR products. This approach, however, is even more stringent on primer design than the Taqman® assay due to the requirement for the presence of at least one guanine on the target sequence for efficient quenching. On the other hand, decrease of fluorescence is not as sensitive and specific due to the presence of heavy fluorescence background in solution.
Thus, there is a long-standing need for the development of primers and systems for use in amplification reactions to improve aspects of primer design and label detection, especially for use in real-time amplification reactions.